Project 5: In addition to adhering to each other and to the vessel wall, platelets contribute to thrombotic[unreadable] events by releasing bioactive molecules such as ADP, TxA2 and CD40L. In studies that form the basis for[unreadable] this proposal, human and mouse platelets were found to express on their surface the class IV semaphorin,[unreadable] sema4D or CD 100, a protein best known for its role in B-cell/T-cell interactions. It was also found that[unreadable] activated platelets shed the exodomain of sema4D and the "sheddase" was identified as the TNFa cleaving[unreadable] enzyme, ADAM17. Based on these observations and preliminary studies on the effects of soluble sema4D on[unreadable] platelets, we have developed the following hypotheses: 1) sema4D, either as a soluble molecule or surfacebound,[unreadable] contributes to platelet activation by binding to receptors expressed on nearby platelets, 2) plateletderived[unreadable] sema4D can also affect cells other than platelets within the circulation and the vessel wall, and 3)[unreadable] plasma levels of soluble sema4D will increase when pathological platelet activation occurs. To test these[unreadable] hypotheses, Aim #1 will examine the role of sema4D in platelet activation. Aim #2 will investigate the[unreadable] regulated shedding of the sema4D extracellular domain. Aim #3 will examine the role of CD72 and plexin-[unreadable] Bl as candidate receptors for platelet-derived sema4D in platelets, monocytes and endothelial cells, and Aim[unreadable] #4 will ask whether platelet activation in vivo causes a measurable increase in plasma sema4D levels that[unreadable] correlates with the extent of platelet activation. Aims #1-3 will take advantage of existing mouse lines[unreadable] lacking sema4D, CD72 or ADAM17. Aim #4 will make use of samples from two clinical trials in which[unreadable] platelet activation is expected. The first trial includes the 1,000 patients undergoing cardiopulmonary bypass[unreadable] in the prospective heparin-induced thrombocytopenia (HIT) trial that is part of Project #1. All of these[unreadable] individuals should have transient platelet activation while on bypass. Those that develop HIT will have[unreadable] persistent platelet activation. The second trial includes 4,000 patients with well-characterized atherosclerotic[unreadable] cardiovascular disease, a setting where platelet activation is predicted to occur, but be less pronounced.[unreadable] Collectively, these aims will address the basic biology of platelet sema4D and its receptors, explore the[unreadable] consequences of sema4D release when platelets are activated, and begin to assess the role of soluble sema4D[unreadable] as a contributor to thrombotic events in vivo.